Epilepsies comprise a remarkably diverse collection of disorders that affect 1–4% of the population in the United States alone. Current therapy is symptomatic. Available drugs reduce seizure frequency in the majority of patients, but it is estimated that only about forty percent are free of seizures despite optimal treatment. From a clinical point of view three types of epilepsy have been defined: (1) petit mal, which is characterized by the absence of seizures or small seizures, (2) grand mal, which comprise generalized catatonic seizures, and (3) complex partial, which is often localized in temporal lobe seizures. The third form is the most common, and it is often resistant to medical treatment. Surgical resection is often the only form of treatment that eliminates seizures in the majority of these resistant patients.
Recombinant adeno-associated virus (rAAV) vectors are useful for gene therapy to the central nervous system (CNS) because these vectors have been found to be non-toxic and have been demonstrated to provide long-term gene expression in the brain (McCown et al., (1996) Brain Res. 713:99) and endogenous gene expression can be controlled using a tetracycline-off, regulatable element (Haberman et al., (1998) Gene Ther. 5:1604).
Focal seizure disorders present an attractive gene therapy target, especially when considering viral vectors as the method of gene delivery. Within the focus, neurons are the cells ultimately responsible for seizure activity, so the ability of viral vectors, such as adeno-associated virus (AAV) and lentivirus, to stably transduce neurons provides access to cells that directly mediate seizure activity (Kaplitt et al., (1994) Nature Genet. 8:148; McCown et al., 1996) Brain Res. 713:99; Naldini et al., (1996) Proc. Nat. Acad. Sci. 93:11382). In addition, there appear to be a number of gene therapy targets that modulate neuronal excitability, such as neurotransmitter receptors and ion channels. In order to pursue these targets, one must consider the opponent nature of inhibitory and excitatory processes in the brain. For example, when the N-methyl-D-aspartic acid (NMDA) excitatory amino acid receptor was targeted with an AAV delivered antisense, focal seizure sensitivity was significantly reduced (Haberman et al., (2002) Mol. Ther. 6:495). However, merely replacing the cytomegalovirus (CMV) promoter with a tetracycline regulated promoter caused the opposite effect, an increase in focal seizure sensitivity. That the two promoters transduced different populations of neurons with only slight overlap suggested an explanation for these seemly contradictory findings. In one case inhibitory interneurons likely comprised the majority of transduced cells, while in the other instance primary excitatory output neurons comprised the preponderance of transduced cells. Therefore, when targeting neurotransmitter receptors or ion channels, the pattern of neuronal tropism can be crucial to the actual physiological outcome.
The adeno-associated viruses (AAV) are members of the family Parvoviridae and the genera Dependoviruses. Serotypes 1 through 4 were originally identified as contaminates of adenovirus preparations (Carter and Laughlin (1984) in, The Parvoviruses p. 67–152 New York, N.Y.) whereas type 5 was isolated from a patient wart that was HPV positive. To date, seven molecular clones have been generated representing the serotypes of AAV (Bantel-Schaal et al. (1999) J. Virol. 73: 939, Chiorini et al. (1999) J. Virol. 73:1309, Muramatsu et al. (1996) Virology 221:208, Rutledge et al. (1998) J. Virol. 72:309, Srivastava et al. (1983) J. Virol. 45:555, Xiao et al. (1999) J. Virol. 73:3994). These clones have provided valuable reagents for studying the molecular biology of serotype specific infection. Transduction of these viruses naturally results in latent infections, with completion of the life cycle generally requiring helper functions not associated with AAV viral gene products. As a result, all of these serotypes are classified as non-pathogenic and believed to share a safety profile similar to the more extensively studied AAV type 2 (Carter and Laughlin (1984) in, The Parvoviruses p. 67–152 New York, N.Y.).
The extensive development of AAV type 2 as a vector has been facilitated by 30 years of studying its biology in vitro. Recombinant AAV type 2 (rAAV2) has proven to be a suitable gene transfer vector in many different organisms (Monohan and Samulski (2000) Gene Ther. 7:24, Rabinowitz and Samulski (1998) Curr. Opin. Biotechnol. 9:470). As the number of applications evaluating gene transfer increases in vitro and in vivo, limitations to efficient rAAV type 2 transduction have become apparent (Bartlett et al. (2000) J. Virol. 74:2777, Davidson et al. (2000) Proc. Natl. Acad. Sci. USA 97:3428, Hansen et al. (2001) J. Virol. 75:4080, Samulski et al. (1999) in, Adeno-associated viral vectors Cold Spring Harbor, N.Y., Walters et al. (2000) J. Virol 74:535, Xiao et al. (1999) J. Virol. 73:3994, Zabner et al. (2000) J. Virol. 74:3852). The natural tropism of any virus, including rAAV type 2, is a fundamental limitation to efficient gene transfer. With the identification of the AAV type 2 receptor, the requirements for efficient entry in target cells have become an active topic of study (Summerford and Samulski (1998) J. Virol. 72:1438). Efforts have been made to overcome these restrictions by broadening the host range using either bispecific antibodies to the virion shell (Bartlett et al. (1999) Nat. Biotechnol. 17:181) or through capsid insertional mutagenesis (international patent publication WO 00/28004; Rabinowitz et al. (1999) Virology 265:274; Girod et al. (1999) Nat. Med. 5:1052, Wu et al. (2000) J.Virol. 74:8635). While these efforts are beginning to bare fruit, utilizing the other serotypes of AAV may yet provide additional resources for making safe and efficient gene transfer vectors. To this end, a number of studies have begun to show the utility of serotype specific vectors in vitro and in vivo (International patent publication WO 00/28004; Chao et al. (2000) Mol. Ther. 2:619, Chao et al., (2001) Mol. Ther. 4:217, Chiorini et al. (3999) J. Virol. 73:1309, Chiorini et al. (1998) Mol. Cell. Biol. 18:5921, Davidson et al. (2000) Proc. Natl. Acad. Sci. USA 97:3428, Hildinger et al. (2001) J. Virol. 75:6199, Xiao et al. (1999) J. Virol. 783:3994, Zabner et al. (2000) J. Virol. 74:3852). In general, each of these studies uncovered broader cell type specificity with increased gene transfer in vivo.
U.S. Pat. No. 6,180,613 describes a method of delivering exogenous DNA to a target cell within the mammalian CNS using a rAAV vector. Haberman et al., (1998) Gene Ther. 5:1604, disclose a dual cassette rAAV vector comprising a reporter gene under the control of a tetracycline-responsive system and the tetracycline transactivator. However, these systems are constrained by the fact that the gene product influences only the cell that has been transduced.
There is an ongoing need in the art for improved methods of nucleic acid delivery to the central nervous system.